1. Field of the Invention
The invention relates to secondary metabolite production by fungi. More particularly, the invention relates to modulation of secondary metabolite production by fungi through genetic manipulation of such fungi.
2. Description of the Related Art
Secondary metabolite production by various fungi has been an extremely important source of a variety of therapeutically significant pharmaceuticals. β-lactam antibacterials such as penicillin and cephalosporin are produced by Penicillium chrysogenum and Acremonium cirysogenum, respectively, and these compounds are by far the most frequently used antibacterials (reviewed in Luengo and Penalva (1994), Prog. Ind. Microbiol. 29: 603–38; Jensen and Demain (1995), Biotechnology 28: 239–68; Brakhage (1998), Microbiol. Mol. Biol. Rev. 62: 547–85). Cyclosporin A, a member of a class of cyclic undecapeptides, is produced by Tolypocladium inflatum. Cyclosporin A dramatically reduces morbidity and increases survival rates in transplant patients (Borel (1986), Prog. Allergy 38: 9–18). In addition, several fungal secondary metabolites are cholesterol lowering drugs, including lovastatin, which is made by Aspergillus terreus and several other fungi (Alberts et al. (1980), Proc. Natl. Acad. Sci. USA 77: 3957–3961). These and many other fungal secondary metabolites have contributed greatly to health care throughout the world (see Demain (1992), Ciba Found. Symp. 171: 3–16; Bentley (1991), Crit. Rev. Biotechnol. 19: 1–40).
Unfortunately, many challenges are encountered between the detection of a secondary metabolite activity to production of significant quantities of pure drug. Thus, efforts have been made to improve the production of secondary metabolites by fungi. Some of these efforts have attempted to improve production by modification of the growth medium or the bioreactor used to carry out the fermentation. Buckland et al. (1989), in Topics in Industrial Microbiology: Novel Microbial Products for Medicine and Agriculture, Elsevier, Amsterdam, pp. 161–169, discloses improved lovastatin production by modification of carbon source and also teaches the superiority of a hydrofoil axialflow impeller in the bioreactor. Other efforts have involved strain improvements, either through re-isolation or random mutagenesis. Agathos et al. (1986), J. Ind. Microbiol. 1: 39–48, teaches that strain improvement and process development together resulted in a ten-fold increase in cyclosporin A production. While important, studies of these types have still left much room for improvement in the production of secondary metabolites.
More recently, strains have been improved by manipulation of the genes encoding the biosynthetic enzymes that catalyze the reactions required for production of secondary metabolites. Penalva et al. (1998), Trends Biotechnol. 16: 483–489 discloses that production strains of P. chrysogenum have increased copy number of the penicillin synthesis structural genes. Other studies have modulated expression of other biosynthetic enzyme-encoding genes, thereby affecting overall metabolism in the fungus. Mingo et al. (1999), J. Biol. Chem. 21: 14545–14550, demonstrate that disruption of phacA, an enzyme in A. nidulans that catalyzes phenylacetate 2-hydroxylation, leads to increased penicillin production, probably by elimination of competition for the substrate phenylacetate. Similarly, disruption of the gene encoding aminoadipate reductase in P. chrysogenum increased penicillin production, presumably by eliminating competition for the substrate alpha-aminoadipate (Casquiero et al. (1999), J. Bacteriol. 181: 1181–1188).
Thus, genetic manipulation holds promise for improving production of secondary metabolites. Genetic manipulation to increase the activity of biosynthetic enzymes for secondary metabolite production or to decrease the activity of competing biosynthetic pathways has proven effective for improving production. Maximum benefit can be achieved by combining several strategies of manipulation. For example, modulating the expression of genes that regulate the biosynthetic enzyme-encoding genes can improve production. In addition, genetic manipulation can be used to impact upon the challenges that are encountered in the fermentor run or downstream processing (e.g., energy cost, specific production of desired metabolite, maximal recovery of metabolite, cost of processing waste from fermentations). There is, therefore, a need for methods for improving secondary metabolite production in a fungus, comprising modulating the expression of a gene involved in regulation of secondary metabolite production in fungi.
One challenge is to identify the types of genes that would be useful for such modulation. Todd and Andrianopoulos (1997), Fungal Genetics and Biology 21: 388–405, teaches that Zn(II)2Cys6 proteins (zinc binuclear cluster proteins, or “ZBC proteins”) are involved in a wide range of processes, including primary and secondary metabolism. This reference teaches that such proteins are primarily, though not exclusively, transcriptional activators. Chang et al. (1995), Applied Environ. Microbiol. 61: 2372–2377, teaches that increased expression of aflR, a ZBC protein, relieves nitrate inhibition of aflatoxin biosynthesis in Aspergillus parasiticus. PCT Publication WO 00/37629, teaches that over-expressing lovE, another ZBC protein, increases lovastatin production in Aspergillus terreus. Noel et al (1998), Mol. Microbiol. 27: 131–142, teaches that xlnR, a ZBC protein, induces expression of xylanolytic extracellular enzymes in Aspergillus niger. Hasper et al. (2000), Mol. Microbiol. 36: 193–200, teaches that xlnR also regulates D-xylose reductase gene expression in Aspergillus niger. D'Alessio and Brandriss (2000), J. Bacteriology 182: 3748–3753, discloses that Gal4p, a ZBC protein, can activate the PUT (proline utilization) genes in a Saccharomyces cerevisiae strain lacking the normal gene for regulation of this pathway, PUT3. PCT Publication WO 00/20596 discloses that prtT, a ZBC protein, activates extracellular proteases in Aspergillus niger. 
Numerous studies have examined the effects of mutations in genes that encode ZBC proteins. Crowley et al (1998), J. Bacteriol. 180: 4177–4183, discloses that a single missense mutation in UPC2, a ZBC gene, results in pleiotropic effects in Saccharomyces cerevisiae. Friden et al. (1989), Mol. Cell. Biol. 9: 4056–4060, teaches that a large internal deletion in Leu3p, a ZBC protein, in Sacclzaromyces cerevisiae causes the protein to be a constitutive transcriptional activator. Oestreicher and Scazzocchio (1995), J. MoL Biol. 249: 693–699, discloses that a single amino acid change in Yc462, a ZBC protein, leads to constitutive, hyperinducible and derepressed expression of at least three genes in Aspergillus nidulans. Wang et al. (1999), J. Biol. Chem. 274: 19017–19024, discloses that nine distinct missense mutations in LEU3 affect the masking of the activation domain of that ZBC protein. Dickson et al. (1990), Nucleic Acids Res. 18: 5213–5217, discloses that single amino acid changes in the C terminal region of Gal4p and Lac9p, two ZBC proteins, lead to constitutive expression of target genes. Marczak and Brandriss (1991), MoL CelL Biol. 11: 2609–2619, teaches that single point mutations in PUT3, a ZBC gene from Saccharomyces cerevisiae, lead to either constitutive or uninducible expression of proline utilization genes. Carvajal et al. (1997), Mol. Gen. Genet. 256: 406–415, teaches that single amino acid substitutions in Pdr1p, a ZBC protein from Saccharomyces cerevisiae, are responsible for over-expression of three transporter genes associated with multiple drug resistance. Nourani et al. (1997), Mol. Gen. Genet. 256: 397–405, teaches that substitutions in a conserved region of Pdr3p, a ZBC protein from Saccharomyces cerevisiae, leads to gain of function mutations. Zhou et al. (1990), Nucleic Acids Res. 18: 291–298, discloses that deletion of all or part of the linker region of Leu3p results in unmodulated activation of Leu3p target genes. Herlich et al. (1998), Fungal Genetics Biol. 23: 1807–1845, teaches that deletion of three amino acids in the C terminus of AflR results in increased expression of the aflatoxin pathway.
These studies demonstrate that ZBC genes can be manipulated in beneficial ways and may have promise as regulators of secondary metabolism. Unfortunately, no one has been able to create a commercial process in which production of a useful secondary metabolite has been significantly increased through the action of a ZBC protein. There is, therefore, a need for new commercial processes using ZBC proteins, or variants thereof, to significantly increase useful secondary metabolite production.